Extraction and quanification of vitamins a &amp; d in fluid samples

ABSTRACT

The invention discloses monoclonal antibodies for vitamins A (retinol palmitate) and D3 (cholecalciferol); a method for using monoclonal antibodies, and the monoclonal antibodies disclosed herein, in particular, to quantitate these vitamins in fluids such as dairy products, and blood, and also raw or processed agri-food and beverage products. The method involves contacting the sample with a mixture of polar and non-polar organic solvents in combination with inorganic salts to remove fat molecules into an organic fraction, and assaying the organic fraction by immunoassay involving the monoclonal antibodies. The presence of a mixture of non-polar and polar organic solvents increases the separation of vitamins from fat molecules and enables the test samples to be quantified by immunoassay without any further treatment.

FIELD OF THE INVENTION

The present invention relates to methods for extracting and quantifyingfat-soluble vitamins from fluid samples. In particular, it relates toantibodies, methods for their use, and kits therefore, to quantifyvitamins A and D in dairy products and in other fluids.

BACKGROUND OF THE INVENTION

Milk which is marketed in Canada and the United States must be fortifiedwith vitamin A and D3 (1,2). Regulatory agencies have set standardsspecifying the minimum amount of vitamins A and D3 to be added to milkproducts. Fortified fluid milk products add value to the agriculturaland dairy industry in that consumers seek products with essentialvitamins and nutrients. Milk processors typically assert general claimsof vitamin fortification levels because current methodology is toocostly and time consuming to implement testing on a batch basis.

Vitamins A and D3 are potentially toxic to humans at higherconcentrations. Since the margin between the nutritionally desirableintake of vitamins and harmful excess is small (3,4,5) it is importantthat errors in fortification levels be detectable in the shortestpossible time. Currently available methodology for these analyses islaborious, tedious, and expensive, and adds to the high cost ofproduction to dairy processors. Currently available methods fordetecting vitamin A and D3 metabolites include binding assays, receptorproteins, high performance liquid chromatography (HPLC), and gaschromatography-mass spectrometry (GC-MS) (6,7,8,9). Such analyses in anylaboratory are time consuming, require skill and expertise on the partof analysts, and require large capital investments for equipment.

Vitamins A and D3 are fat soluble and they are bound by fat molecules ina dairy product such as milk. Accordingly, they must be extracted inlengthy extraction steps. Since vitamins A and D are both labile toheat, light, and oxidation, laborious extraction results in loss of thevitamin in the preparative steps, and often requires 3 to 5 days for thecompletion of the analyses.

Vitamin D plays an active role in the homeostatic mechanism thatcontrols the concentration of calcium ion in plasma. Vitamin D istransported to various sites in the body where it is activated. Theactivated forms of the vitamin act on the target tissues, therebycausing an increase in calcium content. The activation of vitamin D isregulated in a negative feedback system by plasma calcium. The mostbiologically active form of vitamin D is 1,25-dihydroxycholecalciferolor calcitriol, which is formed by two successive hydroxylations ofvitamin D. That is, calcitriol is formed by the sequential hydroxylationof vitamin D at C-25 in the liver and at C-1 in the kidney. Variousother analogs can be produced by hydroxylation at C-24 and C-26. Theabove mentioned biologically active forms of hydroxylated vitamin D aresynthesized in the body only and are not found in dairy or agri-foodproducts. Vitamin D3, or cholecalciferol, is the form used as anadditive and is also produced in the body when the skin, which containsthe provitamin 7-dehydrocholesterol, is exposed to sunlight.

Currently, the principal assay for Vitamin D3 in dairy samples which hasbeen developed is a two step high pressure liquid chromatography (HPLC)assay, whereby the extracted vitamin in about 200 ml of the hexane islyophilized to about 2 ml and the fraction corresponding to vitamin D isisolated by HPLC and collected manually. This collected fraction ofvitamin D is again lyophilized, and dissolved in about 0.5 ml ofmethanol and loaded again on reverse phase HPLC. This assay employs anexpensive and costly laboratory set up and requires trained and skilledpersonnel to handle advanced instrumentation like HPLC (8,9,10).

The principal assays for vitamin A include laborious extractions and theuse of HPLC, or a direct extraction with the detection of vitamin A byspectrofluorometer. The latter process has limitations due to theinterference from other compounds that has fluorescence at the samewavelength. Moreover, because vitamins A and D3 are sensitive toUV-light, they might lose some of their activity due to extensiveextractions, purifications and storage conditions.

The standard assays, which employ one or two step (HPLC) areillustrated, for example, by references (6-9) and (10).

Therefore, there is a need in the art for methods and kits forquantifying vitamins A and D in a fluid sample, and dairy products inparticular.

SUMMARY OF THE INVENTION

This invention discloses methods for quantifying the level of vitamins Aand D3 in fluids, such as dairy products and blood or other bodilyfluids. The methods disclosed herein comprise the extraction of a fluidsample with an extraction solvent to isolate fat-soluble vitamins A andD3 from the rest of the fluid mixture. The vitamins are then quantifiedwith assays which may include using antibodies to vitamins A and D3.Either monoclonal or polyclonal antibodies may be used.

The method would allow dairy processors to test for Vitamins A and D3internally and obtain a result within few hours, so that the percentageof recommended daily amounts of vitamin D contained in the producttested can be indicated directly on the package of milk or other dairyproduct containing the product. In one aspect, the invention comprises amethod of quantifying vitamin A or D3, or vitamin A and D3, in a fluidsample, comprising the steps of:

-   -   (a) extracting the fluid sample with an inorganic salt and an        extraction solvent comprising a mixture of a non-polar organic        solvent and a polar organic solvent, to produce an organic        fraction; and    -   (b) determining the amount of vitamin D3 or vitamin A, or both        vitamin D3 and vitamin A, in the organic fraction.

In one embodiment, the amount of vitamin D3 and/or vitamin A isdetermined using a monoclonal antibody to vitamin D3 and/or vitamin Arespectively. The antibody may be used in a competitive ELISA or asandwich ELISA to quantify the vitamin of interest. Isolated cell linesthat synthesize the monoclonal antibody to vitamin A and vitamin D3 aredescribed herein. In other embodiments, direct measurement assays may beutilized.

In yet another aspect, this invention is a kit comprising a monoclonalantibody, said kit being used to detect vitamin D3 or vitamin A, or bothvitamin D3 and vitamin A in a sample, which may include any fluid suchas those described herein. The kit may include means for quantifying thevitamin which may include assay reagents, glassware and plasticware.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides a method of quantifying fat-solublevitamins, provitamins and their metabolites in a fluid. In oneembodiment, the method is applied to vitamins A and D3 present in adairy product such as milk. Generally, the method comprises the steps ofproviding an antibody that specifically binds to the fat-solublevitamin, which is preferably a monoclonal antibody, extracting thevitamin from the fluid using an extraction solvent, and assaying thevitamin extracted from the fluid.

In this application, the sample fluid is preferably a fluid dairyproduct such as milk, but may also include, without limitation, otherdairy products, an agri-food, a beverage, blood or other biologicalfluids.

In one embodiment, the assay methods of the present invention utilizeantibodies to vitamin D3 and vitamin A, which are preferably monoclonalantibodies. Accordingly, the invention may further comprise thegeneration and purification of a monoclonal antibody against vitamin D3or other forms of vitamin D. The monoclonal antibody which specificallybinds to vitamin D3 does not exhibit significant or substantial bindingactivity to other variants or biologically active forms of vitamin D. Asused herein, vitamin D, also known as calciferol, includes all availableforms of vitamin D including vitamin D3 also known as cholecalciferol.As well, the present invention comprises the generation and purificationof a monoclonal antibody against vitamin A. As used herein, vitamin A,also known as retinol palmitate, includes all active forms of vitamin Aand provitamin A carotenoids. The monoclonal antibody is of coursedistinct from the commercially available polyclonal antibody of vitaminA.

Methods for preparing and isolating polyclonal and monoclonal antibodiesare well known in the art. See, for example, Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; andHurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982. As would beevident to one of ordinary skill in the art, polyclonal antibodies canbe generated from a variety of warm-blooded animals, such as horses,cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with VitaminA or D.

The present invention comprises monoclonal antibodies which specificallybind to vitamin A, as well as monoclonal antibodies which specificallybind to vitamin D3. These monoclonal antibodies are not known in theprior art. In general terms, commercially available pure vitamin A orvitamin D3 may be conjugated to keyhole limpet hemocyanin and used toimmunize mice. Spleen cells may be recovered from the immunized mice andfused with known cell lines. Those hybridomas which produce the desiredmonoclonal antibody may be detected by ELISA. These procedures are wellknown and include standard techniques available to one skilled in theart.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′).sub.2 and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included.

Antibodies are determined to be specifically binding if: 1) they exhibita threshold level of binding activity, and/or 2) they do notsignificantly cross-react with related molecules. Antibodies describedherein specifically bind if they bind to either Vitamin A or Vitamin D3with a binding affinity (K_(a)) of 10⁶ M⁻¹ or greater preferably 10⁷ M⁻¹or greater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹M⁻¹ or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art (Scatchard, G., Ann. NYAcad. Sci. 51: 660-672, 1949).

The methods of the present invention utilize an extraction solvent whichcomprises organic solvents with high selectivity in extracting polarcompounds from fat molecules. In one embodiment, the extraction solventcomprises a polar organic solvent and a non-polar organic solvent incombination with an inorganic salt, to extract fat-soluble compoundsfrom fluids, such as dairy products. As used herein, an “organicsolvent” includes, without limitation, liquid aliphatic hydrocarbonspreferably containing 4 to 10 carbon atoms, or halogenated hydrocarbonscontaining 1 to 4 carbon atoms. Preferred inorganic salts includealkaline metal salts such as potassium hydroxides, chlorides, carbonatesor phosphates. The inorganic salt is preferably dissolved in analcoholic solution, such as a 65% ethanol solution. It is preferred thatthe fluid sample have an alkaline pH for the extraction. The use of apotassium or sodium hydroxide salt of course produces suitable alkalineconditions.

Solvent polarity has been defined and measured in several differentways, one of the most common being the dielectric constant (ε).Aliphatic hydrocarbon solvents typically have dielectric constant valuesless than about 2.0. As used herein, “non-polar” shall refer to anysolvent having a dielectric constant less than 3.0, and preferably lessthan about 2.0. Halogenated hydrocarbon solvents typically havedielectric constants greater than about 8.0. As used herein, “polar”shall refer to any organic solvent having a dielectric constant greaterthan 3.0, and preferably greater than about 4.0, and more preferablygreater than about 8.0.

In one embodiment, the extraction solvent comprises a mixture of hexaneand petroleum ether as the non-polar solvent and methylene chloride(dichloromethane) as the polar solvent. Preferably, equal amounts ofhexane and petroleum ether are mixed with a small amount of methylenechloride. In one embodiment, the solvent comprises hexane, petroleumether and methylene chloride in a 49:49:2 ratio (49:1 non-polar topolar).

Suitable non-polar solvents may include butane, pentane, hexane, heptaneand octane, including mixtures thereof such as petroleum ether, benzeneand acetonitrile, amongst others. Suitable polar organic solvents mayinclude chloroalkanes such as methylene chloride and ethyl acetate.Suitable inorganic salts may include potassium chloride, potassiumhydroxide, or sodium hydroxide, sodium bicarbonate, and sodiumascorbate. It is preferred that the salt raise the pH of the fluidsample to an alkaline pH.

The volume ratio of non-polar to polar in the extraction solvent may begreater than 2:1, and is preferably greater than 5:1, and is mostpreferably greater than about 10:1.

The extraction solvent may be used in ratio of 1:1 or lower with thefluid. In one embodiment, 20 ml of a milk sample may be extracted with15 ml of the preferred extraction solvent described herein. Optionally,an antioxidant such as pyrogallol may be added to the fluid to preventoxidation of the vitamins A and D3 during the extraction procedure.

The mixture of organic solvents in combination with inorganic salts hasthe ability to extract fat-soluble compounds that can be directlydetected by immunoassay without any further treatment. The extractedvitamins can be quantified directly by well-known enzyme-immunoassays,such as by a competitive ELISA, a sandwich ELISA or by radioimmunoassay,which are standard quantitative assays well known in the art (Persoon T.Immunochemical assays in the clinical laboratory. Clinical LaboratoryScience; 5(1): 31-40, 1992).

The extracted vitamins and antibodies can be used for detection andquantification of vitamins A and D3 by direct measurement. The FourierTransform-Infrared Spectroscopy (FT-IR) method is well known and may beapplied through incorporation to biochip technology in the presentinvention. FT-IR is a non-destructive technique that enables theidentification of the unique chemical bonds (a “fingerprint”) of a givenorganic substance, as each chemical bond in a molecule absorbs differentfrequencies at a different wave number. A molecular fingerprint of theprotein/vitamin complex in solution may be obtained, in terms ofinfrared spectra. This spectrum is a unique identity of theprotein/vitamin complex at a given concentration in solution. Otherdirect measurement techniques are well-known in the art.

The methods and antibodies disclosed herein can also be used to preparea kit that can be used to determine the levels of vitamins A and D3 in asample. In one embodiment, the kit comprises an extraction solvent forextraction of the vitamins from the fluid sample, a sample of themonoclonal antibody, and the reagents required for the assay, which maybe an ELISA assay. The kit may additionally comprise hardware, requiredto perform the analysis, such as tubes, assay plates or other glasswareor plasticware.

While the invention has been described in conjunction with the disclosedembodiments, it will be understood that the invention is not intended tobe limited to these embodiments. On the contrary, the invention isintended to cover alternatives, modifications and equivalents, which maybe included within the spirit and scope of the invention as defined bythe appended claims.

The following examples are intended only to illustrate and describe theinvention rather than limit the claims that follow.

EXAMPLES

Materials and Methods

All chemicals were purchased from Sigma Chemical Company (St. Louis,Mo.) unless otherwise noted. Vitamin D3 (Cholecalciferol) was fromMerck. Hexane, petroleum ether, methylene chloride, potassium hydroxidewere from Caledon Laboratories. Bovine serum albumin was from Gibco BRL,and TMB substrate was from KPL Laboratories.

Generation and Identification of Monoclonal Antibodies

1. Conjugation of Retinol Palmitate to Keyhole Limpet Hemocyanin (KLH)

4.1 mg of vitamin A (retinol palmitate) was mixed with KLH and stirredovernight at room temperature. After dialysis, glutaraldehyde was addedto the mixture to a final concentration of 1%. The resulting mixture wasstirred for 6 hrs. The conjugated mixture was dialysed in phosphatebuffered saline (pH 7.4) for 4 hr. Conjugated KLH-vitamin A was filtersterilized and stored in sterile vials at −20° C.

2. Conjugation of Vitamin D3 to Keyhole Limpet Hemocyanin (KLH)

5.4 mg of vitamin D3 was mixed with 10 mg of KLH and stirred overnightat room temperature. After dialysis, glutaraldehyde was added to themixture to a final concentration of 1%. The resulting mixture wasstirred for 6 hrs. The conjugated mixture was dialysed in phosphatebuffered saline (pH 7.4) for 4 hr. Conjugated KLH-vitamin D3 was filtersterilized and stored in sterile vials at −20° C.

3. Immunization and Hybridoma Production for vitamin A

For general procedures related to the generation of monoclonalantibodies, refer to Harlow and Lane, Antibodies—A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. BALB/c mice were immunized, viaintraperitoneal injection, with 50 μg/mouse of the vitamin A-KLHconjugate emulsified in complete Freund's adjuvant. After two weeks, asecond injection of the antigen was given in Freund's incompleteadjuvant, followed by another dose of the antigen in sterile PBS(pH-7.4), after three days. An intrasplenic injection of the antigen wasgiven 10 days following the third dose of antigen. Three days after theintrasplenic injection, spleen cells from the mice were isolated andfused with the Sp2/0-Ag 14 cell line as described by Shulman et al. inNature 276: 269 (1978) using the method of Galfre et al., disclosed inNature 266: 550 (1977). Cells were cultured in 96-well tissue cultureplates in RPMI media supplemented with 20% fetal bovine serum. After 24hrs, selection medium HAT (hypoxanthine, aminopterin and thymidine) wasadded as described by J. W. Littlefield in Science 145: 709 (1964).

4. Elisa with Monoclonal Antibody to Vitamin A

Monoclonal antibodies that bind to vitamin A were detected from culturesupernatants by using ELISA. Microtitre plates Falcon) were coated withretinol palmitate (20 ug/ml in PBS, pH 7.4) and incubated at 4° C.overnight. Plates were washed with PBS (pH 7.4) and blocked with 3%bovine serum albumin for 3 hrs. Plates were washed again with PBS, andair-dried. Wells exhibiting hybridoma growth were marked and thesupernatant (100 ul) from each well was transferred to the vitamin Acoated plate and incubated overnight at 4° C. Commercially availablepolyclonal antibody for vitamin A, and anti-sera collected as blood fromtail vein of the mice after the third intraperitoneal injection ofvitamin A (positive control representing the high titre antibody againstvitamin A), were run concurrently as controls. Pre-bleed sera (collectedas blood from tail vein of the mice before the vitamin A injection) werealso run concurrently as negative control. Bound monoclonal antibody wasdetected with enzyme-labeled antibody (1:6,000 dilution, Sigma) usingTMB as substrate.

Cells from positive wells were transferred to 24-well plates followed by6-well plates and then into T25 tissue culture flasks. The cell linesproducing high titre of antibody are SM-1.4A and SM-4.12A.

5. Immunization and Hybridoma Production for Vitamin D3

For general procedures related to the generation of monoclonalantibodies, refer to Harlow and Lane, Antibodies—A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. BALB/c mice were immunized, viaintraperitoneal injection, with 50 μg/mouse of the vitamin D-KLHconjugate emulsified in complete Freund's adjuvant. After two weeks, asecond injection of the antigen was given in Freund's incompleteadjuvant, followed by another dose of the antigen in sterile PBS(pH-7.2), after three days. An intrasplenic injection of the antigen wasgiven 10 days following the third dose of antigen. Three days after theintrasplenic injection, spleen cells from the mice were isolated andfused with the Sp2/0-Ag 14 cell line as described by Shulman et al. inNature 276: 269 (1978) using the method of Galfre et al., disclosed inNature 266: 550 (1977). Cells were cultured in 96-well tissue cultureplates in RPMI media supplemented with 20% fetal bovine serum.

After 24 hrs, selection medium HAT (hypoxanthine, aminopterin andthymidine) was added as described by J. W. Littlefield in Science 145:709 (1964).

6. ELISA with Monoclonal Antibody to Vitamin D3

Monoclonal antibodies that bind to vitamin D3 were detected from culturesupernatants by using ELISA. Microtitre plates (Falcon) were coated withvitamin D3 (20 ug/ml in PBS, pH 7.4) and incubated at 4° C. overnight.Plates were washed with PBS (pH 7.4) and blocked with 3% bovine serumalbumin for 3 hrs. Plates were washed again with PBS, and air-dried.Wells exhibiting hybridoma growth were marked and the supernatant (100ul) from each well was transferred to the vitamin D3 coated plate andincubated overnight at 4° C. Commercially available monoclonal antibodyfor 1-25 dihydroxy vitamin D3 and anti-sera collected as blood from tailvein of the mice after the third intraperitoneal injection of vitamin D3(positive control representing a high titre antibody against vitaminD3), were run concurrently as positive controls. Pre-bleed sera(collected as blood from tail vein of the mice before the vitamin D3injection) were also run concurrently as negative control. Boundmonoclonal antibody was detected with enzyme-labeled antibody (1:6,000dilution, Sigma) using TMB as substrate.

Cells from positive wells were transferred to 24-well plates, followedby 6-well plates and then into T25 tissue culture flasks. The cell linesproducing high titre of antibody are SM 4.9A, SM 4.9B and SM 1.1E.

Extraction of Vitamins A and D3 from Milk

20 ml of well-mixed fluid milk, at room temperature, was placed intoamber colored bottle, followed by addition of 2 ml of 10% pyrogallolantioxidant, and mixed for 5 min. Then slowly from the sides of thebottle, cold alcoholic potassium hydroxide solution was added to achievea ratio of potassium, ethanol and water of 1:2:0.3. Samples were placedin the incubator, and extracted with 15 ml of a mixture of organicsolvents. The solvent mixture comprised hexane: petroleum ether:methylene chloride, in a ratio of 49:49:2. The sample was then eithercentrifuged or placed on the table for 10 min, to separate the phases.The supernatant may be used directly for immunoassay for thequantification of vitamins A & D3.

Quantification of Vitamin A and D3 in Dairy Samples Using ELISA

Conventional assays utilizing monoclonal or polyclonal antibodies forvitamin D (including its metabolites and analogs) and vitamin A(including precursors or provitamins, metabolites and analogs) are wellknown in the art. Such assays include competitive binding assays andenzyme-linked immunoassays which are well known in the art. For example,methods to assay for 1,25-dihydroxyvitamin D are described (11) in Chenet al., J. Nutr. Biochem. 1:320-327 (1990), and in U.S. Pat. Nos.4,297,289, 4,816,417, 4,585,741 and 5,232,836.

REFERENCES

The following references are cited in the application as numbers inbrackets [( )] at the relevant portion of the application. In addition,there are references cited within this application. Each of thesereferences, whether cited in the body of this application or below, isincorporated herein by reference.

-   1. The Food and Drugs Act and Regulations; Department of National    Health and Welfare, Section B.08.003 to B.08.006; Jul. 10, 1991-   2. Grade “A” Pasteurized Milk Ordinance, U.S. Department of Health    and Human Services, Public Health Service; Food and Drug    Administration; 1995 Revision-   3. Kirschmann, G & J, Nutrition Almanac 4^(th) ed. New York: McGraw    Hill, 1996-   4. Reinhold Veith, Vitamin D Supplementation, 25-hydroxyvitamin D    concentrations, and safety 1, 2; American Journal of Clinical    Nutrition, Vol. 69, No. 5, 842-856, May 1999-   5. Blank, S., Scanlon, K. S., Sinks, T. H., Let, S., and Falk, H. An    outbreak of hypervitaminosis D associated with the overfortification    of milk from a home delivary dairy. American J. Publ. Health, 855,    656-659, 1995-   6. Silva, MG, and Sanders J K, Vitamin D in Infant Formula and    Enteral Products by Liquid Chromatography: Collaborative Study, J.    AOAC Int., January-February 1996; 79 (1): 73-80-   7. Kurmann, A, and Indyk, H, Endogenous Vitamin D Content of Bovine    Milk: Influence of Season, Food Chem., 50 (1): 75-81, 1994-   8. Agarwal, V K, Liquid Chromatographic Determination of Vitamin D    in Animal Feeds and Premixes, J. AOAC Int., 75: (5): 812-815, 1990-   9. Vitamin D in fortified milk and milk powder; Liquid    chromatographic method; AOAC Official Methods of Analysis; 15^(th)    ed., 1068-1069, 1990-   10. Method for the analysis of vitamins A & D in milk, U.S.    Department of Health and Human Services, Public Health Service; Food    and Drug Administration—LQAB A & D; 1995 Revision-   11. Chen, T. C., Turner, A. K., and Holick, M. F. A method for the    determination of circulating concentration of 1,25-dihydroxyvitamin    D, J. Nutr. Biochem., 1, 1990.

1. A method of quantifying vitamin A or D3, or vitamin A and D3, in afluid sample, comprising the steps of: (a) extracting the fluid samplewith an inorganic salt and an extraction solvent comprising a mixture ofa non-polar organic solvent and a polar organic solvent, to produce anorganic fraction; and (b) determining the amount of vitamin D3 orvitamin A, or both vitamin D3 and vitamin A, in the organic fraction. 2.The method of claim 1 wherein the vitamin is quantified with an antibodywhich specifically binds to the vitamin.
 3. The method of claim 2wherein the antibody is a monoclonal antibody.
 4. The method of claim 2wherein the antibody is a polyclonal antibody.
 5. The method of claim 1wherein the non-polar organic solvent comprises an aliphatic hydrocarbonhaving from 4 to 10 carbon atoms, or a mixture thereof, and the polarorganic solvent comprises a halogenated hydrocarbon having from 1 to 4carbon atoms, or a mixture thereof.
 6. The method of claim 1 wherein theinorganic salt is an alkaline metal salt.
 7. The method of claim 1wherein the inorganic salt is added to the sample in an alcoholicsolution.
 8. The method of claim 7 wherein the alcoholic solutioncomprises greater than about 50% and less than about 75% ethanol inwater (v:v).
 9. The method of claim 6 wherein the alkaline metal saltcomprises potassium hydroxide.
 10. The method of claim 5 wherein thenon-polar organic solvent comprises a mixture of hexane and petroleumether and the polar organic solvent comprises methylene chloride
 11. Themethod of claim 1, wherein the fluid sample is chosen from the groupconsisting of a dairy product, an agri-food, a beverage, blood or abiological fluid.
 12. The method of claim 1 wherein the vitamin D3 orvitamin A, or vitamins D3 and vitamin A, is detected by an immunosorbentassay.
 13. The method of claim 1 wherein the vitamin is quantified bydirect measurement.
 14. A monoclonal antibody which specifically bindsto vitamin D3 and not to its metabolites or other forms of vitamin D.15. An isolated cell line which produces the antibody of claim
 14. 16.(Cancelled).
 17. An isolated cell line which produces the antibody ofclaim
 16. 18. A kit for assaying for vitamin D3 or vitamin A, or vitaminD3 and vitamin A, in a fluid sample comprising: (a) an inorganic saltand an extraction solvent comprising a mixture of a non-polar organicsolvent and a polar organic solvent, to produce an organic fraction, (b)a monoclonal antibody which specifically binds to vitamin D3 or vitaminA, and (c) means for performing a quantitative assay with the antibody.19. The kit of claim 17 wherein the means for performing a quantitativeassay comprises reagents and hardware for a competitive ELISA or asandwich ELISA.